The sphingolipid ceramide is now well established as an endogenous regulator of apoptosis in many different cancer cell systems, and in response to many cytotoxic and chemotherapeutic agents. Several targets of ceramide action have been identified, and its relationship to several components of the apoptotic pathway has begun to be deciphered. However, the exact mechanism of ceramide action and its potential as a chemotherapeutic agent have yet to be defined. To this end we attempted to increase endogenous ceramide levels in cancer cells by developing Bacillus cereus sphingomyelinase as a tool to hydrolyze sphingomyelin for mechanistic and future chemotherapeutic studies. Exogenous treatment with bSMase increased ceramide levels but did not induce apoptosis, whereas, endogenous expression of bSMase induced apoptosis. Because of this apparent compartmental difference in cellular ceramide, we elected to target bSMase to different cellular compartments. Our data demonstrate that only when bSMase was targeted to mitochondria did cells undergo apoptosis, whereas its targeting to cytosol, ER or nucleus was ineffective. This was despite increases in ceramide levels in all the compartments. These exciting data led us to propose the following hypothesis: ceramide is generated in mitochondria in response to inducers of apoptosis and it mediates its biologic effects through mitochondrial processes. Modulating ceramide levels in mitochondria may have novel therapeutic implications for cancer treatment. In this proposal our goals are the following: 1) determine if generation of ceramide in mitochondria is sufficient to induce apoptosis, 2) determine the mechanism by which mitochondrial ceramide activates the apoptotic program, 3) determine if mitochondrial ceramide generation is necessary to activate apoptosis in response to chemotherapeutic agents, and 4) determine if modulating mitochondrial sphingolipids has therapeutic implications for cancer treatment. These studies should determine if ceramide is necessary and sufficient for induction of mitochondrial processes of apoptosis, decipher specific mitochondrial targets and mechanisms of ceramide action, and allow us to begin exciting studies to develop mitochondrial ceramide as a specific therapeutic inducer of cancer cell death.